Serum concentrations of IgM, IgG1, IgG2b, IgG3 and IgA in C57BL/6 mice and their congenics at the lpr (lymphoproliferation) locus

The serum concentrations of IgM, IgG1, IgG2b, IgG3 and IgA were determined in mice of C57BL/6 background, from weaning to one year of age, by quantitative isotype-specific, indirect double sandwich enzyme-linked immunosorbent assays (ELISAs). Only limited data could be obtained for the IgG2a isotype in the present study. The mean serum Ig levels found for 6-month-old B6 mice were 0.22 mg/ml for IgM, 0.28 mg/ml for IgG1, 1.22 mg/ml for IgG2b, 0.18 mg/ml for IgG3, 0.075 mg/ml for IgA and about 0.7 mg/ml for IgG2a. In comparison with mice of the wild strain, C57BL/6 mice homozygous at the lpr (lymphoproliferation) locus showed very high increases in serum Ig levels when older than 20 weeks. With 6-month-old B6 lpr mice, increases in concentration were found for all tested heavy chain isotypes: 6 to 6.5-fold for IgA (0.45 mg/ml) and IgG1 (1.82 mg/ml), 9-fold for IgG3 (1.6 mg/ml), 11 to 11.5-fold for IgM (2.44 mg/ml) and IgG2b (13.8 mg/ml) and about 8-fold for IgG2a (5.5 mg/ml). Therefore homozygosity at the lpr locus provides the conditions for generalized, poly-isotypic rather than isotype-specific restricted Ig enhancement. This observation may be more compatible with hyperinducibility of all B-cell subclasses than with excessive production of T-cell-derived factors whose activity would be expected to be restricted to some T-dependent subclasses, and at least to affect IgM-committed B cells to a lesser extent than other B-cell classes.     

Serum concentrations of IgM, IgG1, IgG2b, IgG3 and IgA in C57BL/6 mice and their congenics at the lpr (lymphoproliferation) locus

The serum concentrations of IgM, IgG1, IgG2b, IgG3 and IgA were determined in mice of C57BL/6 background, from weaning to one year of age, by quantitative isotype-specific, indirect double sandwich enzyme-linked immunosorbent assays (ELISAs). Only limited data could be obtained for the IgG2a isotype in the present study. The mean serum Ig levels found for 6-month-old B6 mice were 0.22 mg/ml for IgM, 0.28 mg/ml for IgG1, 1.22 mg/ml for IgG2b, 0.18 mg/ml for IgG3, 0.075 mg/ml for IgA and about 0.7 mg/ml for IgG2a.In comparison with mice of the wild strain, C57BL/6 mice homozygous at the lpr (lymphoproliferation) locus showed very high increases in serum Ig levels when older than 20 weeks. With 6-month-old B6 lpr mice, increases in concentration were found for all tested heavy chain isotypes: 6 to 6.5-fold for IgA (0.45 mg/ml) and IgG1 (1.82 mg/ml), 9-fold for IgG3 (1.6 mg/ml), 11 to 11.5-fold for IgM (2.44 mg/ml) and IgG2b (13.8 mg/ml) and about 8-fold for IgG2a (5.5 mg/ml). Therefore homozygosity at the lpr locus provides the conditions for generalized, poly-isotypic rather than isotype-specific restricted Ig enhancement. This observation may be more compatible with hyperinducibility of all B-cell subclasses than with excessive production of T-cell-derived factors whose activity would be expected to be restricted to some T-dependent subclasses, and at least to affect IgM-committed B cells to a lesser extent than other B-cell classes.     

Comparative patterns of serum immunoglobulin levels in specific-pathogen-free congenitally athymic (nude), hereditarily asplenic (Dh/+), congenitally athymic-asplenic (lasat) and splenectomized athymic mice.

Serial determinations of serum immunoglobulin levels were assessed in congenitally athymic (nude), hereditarily asplenic (Dh/+) and congenitally athymic-asplenic (lasat) mice and the results compared to normal intact littermate controls (nu/+), neonatally splenectomized nu/+ and neonatally splenectomized nude mice. Quantification of Ig levels was accomplished by radial immunodiffusion, for IgM, IgG1, IgG2a, IgG2b and IgA antibody isotypes. Intact spleen and/or thymus function was shown to have marked effects on the age-dependent development of serum IgM, IgG2b and IgA production. Furthermore, because of higher levels of IgA in congenitally athymic-asplenic mice and neonatally splenectomized nude mice v. sham splenectomized nude mice, it is suggested that an IgA-specific suppressor population resides in the spleen. Finally, because of frequent problems in the literature in interpretation of immunoglobulin values, the criteria for the statistical evaluation of such data in establishing normal serum Ig values and ascertaining real differences between treatment groups are emphasized.     

Immunoglobulin isotypes of C57BL/6 nu/nu, lpr/lpr mice. Lack of direct intrinsic effect of the lpr gene on B cell hyperactivity

The absolute concentrations of mouse immunoglobulin (Ig) heavy chain isotypes were determined by specific ELISAs in the serum of C57BL/6 (B6) mice doubly homozygous at the nude (nu) and the lymphoproliferation (lpr) locus (B6 nu, lpr mice), and compared with normal B6 nu mice. The distribution and the absolute concentrations of all Ig isotypes were found to be very similar in B6 nu, lpr and B6 nu mice, for both sexes and with similar increases in titers with ageing. Thus, the major part of the severe autoimmunity and hyperglobulinemia characteristic of the lpr syndrome of euthymic B6 lpr mice, including their elevated titers of thymus-independent IgM and IgG3 isotypes, is abrogated by the nude mutation, an effect of which is the lack of thymus differentiation. Though a postulated intrinsic activity of the lpr gene directly on B cell hyperactivity cannot be discarded, its expression would then require the presence of either the thymus or of T cells or of other cells or factors whose expression is also abrogated by the homozygosity at the nude locus.     

Serum immunoglobulins in nude mice and their heterozygous littermates during ageing.

Serum immunoglobulin (Ig) levels were investigated in 6, 40 and 110 week old congenitally athymic (nude) mice and their heterozygous littermates. Concentrations of IgM, IgG1, IgG2a, IgG2b, IgG3 and IgA were determined by rocket electrophoresis. At 6 weeks of age, IgM was the most prominent serum Ig in both nude and heterozygous mice. Except for IgM and IgG3, some nude mice displayed unquantifiable levels of some of the other Ig classes or subclasses. At this age, the average levels of the various Ig classes and/or subclasses did not differ significantly between the two groups of mice. At the ages of 40 and 110 weeks, most nude mice showed serum Ig spectra in which all classes and subclasses were present. Young (6 week) and middle-aged (40 week) nude mice generally showed a wider variation in Ig levels than did their heterozygous littermates. The most striking differences between aged nude mice and aged heterozygous mice were: (a) the generally decreased levels of IgG2a, IgG2b and IgA; (b) the frequent occurrence of increased serum levels of IgG1; and (c) the increased incidence of homogeneous Ig components (''paraproteins'') in the sera of nude mice.     

Immunoglobulins of rnu/rnu ''nude'' rats with congenital aplasia of the thymus

The levels of immunoglobulins (Ig) in the serum and saliva of nude (rnu/rnu) congenitally athymic rats and control (rnu/+) thymic heterozygous rats have been studied. Serum IgM, and serum and salivary IgA levels were virtually identical between rnu/rnu and rnu/+ rats. Serum IgG was drastically reduced in nude rats during the period when endogenous IgG synthesis was predominant. The most consistent and pronounced differences in Ig levels between rnu/rnu and rnu/+ rats involved the IgG isotype.     

Evidence of Thymus-Independent Local and Systemic Antibody Responses to Cryptosporidium parvum Infection in Nude Mice

Differences in susceptibility to persistent cryptosporidial infection between two strains of adult athymic nude mice prompted us to investigate the immune mechanism(s) that may control resistance to infection in these T-cell-deficient mice. We studied fecal oocyst shedding, serum and fecal parasite-specific antibody responses, and fecal immunoglobulin levels in athymic C57BL/6J nude and athymic BALB/cJ nude mice following oral inoculation with Cryptosporidium parvum oocysts at 8 to 9 weeks of age. C57BL/6J nude mice had significantly higher fecal parasite-specific immunoglobulin A (IgA) (days 27, 31, 35, and 42 postinoculation) and IgM (days 10, 17, 24, 28, 31, 38, 42, and 48 postinoculation) levels than BALB/cJ nude mice (P < 0.05) and significantly higher serum parasite-specific IgA levels at 63 days postinoculation (P < 0.03). Moreover, C57BL/6J nude mice shed significantly fewer C. parvum oocysts than BALB/cJ nude mice from days 52 to 63 postinoculation (P < 0.05). In contrast, BALB/cJ nude mice had higher levels of non-parasite-specific IgA (days 38 to 63 postinoculation) and IgM (days 24, 35, 38, and 52 postinoculation) than C57BL/6J nude mice in feces (P < 0.05). These data suggest that parasite-specific fecal antibodies may be associated with resistance to C. parvum in C57BL/6J nude mice.     

CD4+ T cells play a major role for IgM and IgG anti-DNA production in mice infected with Plasmodium yoelii

The infection by a non-lethal strain of Plasmodium yoelii induces the formation of autoantibodies such as anti-DNA and anti-Sm antibodies in mice. The extent of the relative increase in serum levels of IgM and IgG anti-DNA and anti-Sm antibodies and their kinetics were found to be similar to those of anti-hapten antibodies and of total IgM and IgG levels. This strongly suggested that anti-DNA and anti-Sm autoantibody responses observed in malaria-infected mice are a result of polyclonal activation of B cells. The analysis of the IgG subclasses reacting with DNA antigen showed significant levels of the T cell-dependent isotypes, IgG1 and IgG2. The role of T cells in the activation of autoreactive B cells was confirmed by using athymic nude mice. Indeed, BALB/c-nu/nu and C57BL/6-nu/nu mice failed to produce IgG anti-DNA antibodies after infection with P. yoelii. Moreover, the reconstitution of BALB/c nude mice with lymph node cells from congenic euthymic BALB-Igb mice showed the activation of autoreactive B cells in nude mice by T cells from euthymic mice. Studies in mice depleted of CD4+ T cells strongly suggested that malaria-induced anti-DNA antibodies were almost entirely dependent on the presence of CD4+ T cells, as this depletion significantly decreased IgM anti-DNA antibodies and completely abolished the IgG anti-DNA production, including the IgG3 subclass in infected mice. In contrast, depletion of the CD8+ T cell subset had no effect on the production of autoantibody in malaria-infected mice. Our results indicate that CD4+ T cells play a major role for both IgM and IgG anti-DNA production during the course of malaria infection.     

Indirect double sandwich ELISA for the specific and quantitative measurement of mouse IgM, IgA and IgG subclasses

We have developed sensitive enzyme-linked immunosorbent assays (ELISA) which measure mouse serum heavy chain immunoglobulin isotypes in nanograms per milliliter. In each case the specific isotypic Ig is sandwiched between an isotype-specific antibody used for coating and another isotype-specific antibody coupled to biotin for detection (with alkaline phosphatase coupled to avidin). These methods are simple to perform, specific for each isotype, reproducible with an average coefficient of variation of 5% for IgG1, 3% for IgG2a, 7% for IgG2b, 10% for IgG3, 3% for IgA and 7% for IgM, and at least 100 times more sensitive than radial immunodiffusion. The assays have been used to determine the absolute concentrations of mouse serum heavy chain Ig isotypes.     

Distribution of IgM, IgA and IgG secreting cells in the tissues of normal and tumour-bearing mice.

The levels of IgM, IgA and IgG secreting cells were examined in control, Corynebacterium parvum-stimulated and tumour-bearing, normal and athymic (Nu/Nu) mice. The percentage of IgA to IgM or IgG secreting cells is relatively higher in peripheral blood than in the spleen or peritoneum of normal mice. Within tumours, irrespective of their degree of vascularization and immunogenicity, the pattern of Ig secreting cells in similar to that seen in peripheral blood and different from that in spleen and peritoneum even in athymic mice. Intraperitoneal injection of C. parvum changes the relative percentages of Ig secreting cells in the peritoneal cavity to resemble that seen in the peripheral blood and tumours. It appears that Ig secreting cells extravasate from peripheral blood in a non-isotype specific manner into sites of chronic stimulation.     

T cell dependent differentiation of human B cells: direct switch from IgM to IgE, and sequential switch from IgM via IgG to IgA production.

Ig production by splenic human B cells that express different surface Ig isotypes were analysed in limiting dilution cultures. Therefore, FACS sorted IgM+, IgG+ and IgA1+ B cells were stimulated with PMA-activated EL4 thymoma cells as helper cells in the presence of IL-2 and IL-4. We found that at least every second B cell responded in vitro and secreted the antibody corresponding to its surface Ig isotype. IgE secreting cells developed from surface IgM+ D+ cells (1/31 to 1/167), but not from IgG+ or IgA1+ cells (much less than 1/5000). Negative signalling of the IgM+ B cells by addition of anti-IgM antibodies into the cultures reduced the number of single IgM producing cells by greater than 85%, and completely inhibited IgE switch. In contrast, anti-IgG and anti-IgA antibodies did not reduce the IgE response. The results indicate a direct switch from IgM to IgE secretion in vitro. In contrast to IgE, IgA secreting cells developed from IgM+D+ (1/30 to 1/51) and from IgG+ B cells (1/14 to 1/25). Negative signalling of the IgG+ B cell subset within total B cells by anti-IgG antibodies suppressed the development of IgG as well as IgA producing cells, but did not inhibit IgM and IgE responses. This indicates a sequential switch from IgM via IgG to IgA. Taken together, this study indicates that IgE secreting cells are derived directly from IgM+D+ B cells by non-sequential switching, whereas IgA producing cells preferentially develop by sequential switching via IgG+ B cells.     

Heterogeneity of Serum IgG Subclass Deficiencies in B Chronic Lymphocytic Leukemia

The occurrence of abnormally low serum immunoglobulin (Ig) levels is well-known in B chronic lymphocytic leukemia (CLL), but published data on IgG subclass levels are virtually absent. We measured serum IgG subclass levels in 52 B CLL outpatients, most in stage A and untreated, using an indirect immunoenzymatic assay with monoclonal antibodies. Mean levels of all Ig isotypes were lower than in normal controls in the whole group of patients, except for IgG2 in those studied at diagnosis. Levels of IgG1, IgG2, IgA, and IgM were lower in patients with a long disease duration than in those studied earlier. IgG subclass deficiencies occurred in 54% of cases and the most frequently affected isotype was IgG1. Every possible combination of IgG subclass and Ig class deficiencies from the selective deficiency of a single subclass to a combined deficiency of all isotypes was observed. This marked heterogeneity argues against the occurrence of isolated defects of one of the cytokines involved in Ig switching as a cause of hypoimmunoglobulinemia in CLL.     

Quantification of turkey immunoglobulins IgA, IgM and IgG in serum and secretions

The immunoglobulins IgA, IgM and IgG of the turkey were quantitated in individual serum samples as well as in pooled sera. The variability of Ig levels in normal, healthy birds was quite high: IgA: mean value 0.633 mg/ml (4.0 -x - 2.5 -x); IgM: mean value 4.37 mg/ml (0.5 -x - 1.4 -x) and IgG: mean value 8.92 mg/ml (0.6 -x - 1.7 -x). Immunoglobulin levels in egg-yolk, egg-white, bile and some intraocular tissues were quantitated as well. An interesting finding was, that the forementioned variability was by far not so high with respect to IgG levels in 20 egg-yolk samples: mean value 5.1 mg/ml (0.86 x- - 1.17 -x). Though IgG and IgM could be detected in pooled turkey bile, IgA predominated in this secretion. In aqueous humor, iris tissue and vitreous body only IgG could be detected.     

Liver Damage in Mice and Rats Causes Tenfold Increase of Blood Immunoglobulin A

Average serum concentrations of IgA and IgM of the SDK rats were 0.27 and 0.43 mg/ml, respectively. Subtotal hepatectomy had no detectable effect on the concentration of IgM but IgA increased in 4 days to a level of 4.9 rag/ml. Thereafter the concentration fell back to normal in 21 days. Serum concentrations of IgA, IgM and IgG2a in (C57BL × CBA)F₁ mice were 0.41, 0.35 and 1.67 mg/ml, respectively. Neither hepatectomy nor carbon tetrachloride poisoning had an effect on the concentrations of IgM or IgG2a but IgA increased to 3.0 mg/ml (hepatectomy) or 3.6 mg/ml (carbon tetrachloride poisoning). Again the increase was reversible. IgA of normal rat serum was mostly monomeric (7S) and of normal mouse serum a mixture 7S and 9S (dimeric) IgA. After liver damage of either type the majority of the serum IgA had a sedimentation constant higher than 7S. It thus resembled the IgA that is brought to the circulation by the thoracic duct of rats and mice.     

Serum immunoglobulin isotype profile of viable and non viable lymphoid cell chimaeras made with nude athymic lpr (lymphoproliferation) mouse recipients.

C57BL/6 mice (B6) which are homozygous at the nu (nude, athymic) and lpr (lymphoproliferation) locus (B6 nulpr) are short-lived. We showed previously that increased survival could be obtained by grafting lymphoid cells from euthymic lpr-homozygous B6 mice (B6 lpr) mice ([lpr----nulpr] chimaeras), but curiously enough not from normal (B6 wild) mice ([wild----nulpr] chimaeras). Moreover female, but not male, [lpr----nulpr] chimaeras developed spleen and lymph node enlargement. In the present paper the distribution and absolute concentrations of all serum immunoglobulin (Ig) isotypes have been determined in these chimaeras and their controls. All chimaeras displayed whole serum Ig levels higher than those of B6 wild mice, suggesting a successful reconstitution of the athymic recipients by the grafted lymphoid cells, but two types of chimaeras were peculiar. The short-lived [wild----nulpr] chimaeras showed a proportion of IgM as high as ungrafted B6 nulpr mice, suggesting a deficient down-regulation of IgM production by the grafted B6 wild-type lymphoid cells. The [lpr----nulpr] female chimaeras recovered a long lasting overexpression of all Ig isotypes, like B6 lpr mice, while all the other chimaeras showed a transient overexpression only. Since neither lymphadenopathy nor persistent increase of serum Ig levels were observed in [lpr----nu] chimaeras, our data confirmed the need for a genetically lpr host to allow the significant development of the lpr syndrome.     

Immunoglobulin levels of congenitally athymic rats immunized with thymus-dependent-independent antigens.

The levels of immunoglobulins (Ig) in the serum and saliva of nude (rnu/rnu), congenitally athymic rats and control (rnu/+) rats were studied after injection in the vicinity of the salivary glands with several different T-dependent or T-independent dinitrophenylated antigens. Serum IgG levels were higher in rnu/+ rats than rnu/rnu rats after both primary and secondary immunizations. WHile serum IgM levels were higher in rnu/+ rats after primary immunization, secondary immunization with the most thymic-dependent antigens elicited higher IgM levels in rnu/rnu rats. After primary immunization, serum IgA levels in rnu/rnu rats were significantly lower than rnu/+ levels but no differences between the groups were noted after secondary immunization. Primary immunization with these antigens also demonstrated a markedly reduced salivary IgA producing capability by the athymic rats. Importantly, no such differences in serum or salivary IgA were previously found in the absence of immunization. Thus, immunization accentuated differences in Ig level and isotype distribution between rnu/rnu and rnu/+ rats.     

Antibody activity to herpes simplex virus in mouse Ig classes and IgG subclasses

Summary Recent studies indicate that Ig class and IgG subclass induction varies for different proteins and further that some Ig subclasses, like IgG2a, are more efficient in important biologic processes such as antibody-dependant cell-mediated cytotoxicity (ADCC). Many proteins of herpes simplex virus type 1 (HSV-1) are immunogenic and induce immunoglobulin responses. To determine the distribution of immunoglobulins induced by HSV-1 proteins, we studied immune mouse serum using an Ig isotype specific Elisa assay for antiviral activity. We found by endpoint analysis that the antiviral titer was 1:12,903 for IgG1, 1:5141 for IgG2a, 1:2140 for IgG2b and 1:229 for IgG3. To identify which isotypes were induced by individual glycoproteins and other viral proteins, Western blots containing HSV-1 proteins were probed with immune serum and isotype specific second antibodies. gB, gC, gD and the 42/44KDa nucleocapsid complex induced strong IgG1, IgG2a, IgG2b responses. IgG3 reactivity with viral proteins appeared weaker. Among the IgG3 reactivities detected on immunoblots, gB and gC were the most intense. Other proteins which elicited IgG1, IgG2a and IgG2b responses were 170KDa, 154KDa and gE. IgA responses were induced by 154KDa, gC, gB, gE and gD. Prominent IgM responses included gB, gC, gD and the 42KDa protein. These results indicate that HSV-1 glycoproteins induce prominent responses in all IgG isotypes except IgG3. The biologic implications of the data are discussed.     

Preferential production of IgG1, IL-4 and IL-10 in MuSK-immunized mice

Myasthenia gravis (MG) is an autoimmune disease characterized by muscle weakness associated with acetylcholine receptor (AChR), muscle-specific receptor kinase (MuSK) or low-density lipoprotein receptor-related protein 4 (LRP4)-antibodies. MuSK-antibodies are predominantly of the non-complement fixing IgG4 isotype. The MuSK associated experimental autoimmune myasthenia gravis (EAMG) model was established in mice to investigate immunoglobulin (Ig) and cytokine responses related with MuSK immunity. C57BL/6 (B6) mice immunized with 30 μg of recombinant human MuSK in incomplete or complete Freund's adjuvant (CFA) showed significant EAMG susceptibility (> 80% incidence). Although mice immunized with 10 μg of MuSK had lower EAMG incidence (14.3%), serum MuSK-antibody levels were comparable to mice immunized with 30 μg MuSK. While MuSK immunization stimulated production of all antibody isotypes, non-complement fixing IgG1 was the dominant anti-MuSK Ig isotype in both sera and neuromuscular junctions. Moreover, MuSK immunized IgG1 knockout mice showed very low serum MuSK-antibody levels. Sera and MuSK-stimulated lymph node cell supernatants of MuSK immunized mice showed significantly higher levels of IL-4 and IL-10 (but not IFN-γ and IL-12), than those of CFA immunized mice. Our results suggest that through activation of Th2-type cells, anti-MuSK immunity promotes production of IL-4, which in turn activates anti-MuSK IgG1, the mouse analog of human IgG4. These findings might provide clues for the pathogenesis of other IgG4-related diseases as well as development of disease specific treatment methods (e.g. specific IgG4 inhibitors) for MuSK-related MG.     

Antibody and cytokine responses to the cilium-associated respiratory bacillus in BALB/c and C57BL/6 mice

The cilium-associated respiratory (CAR) bacillus is a gram-negative, gliding bacterium that causes persistent respiratory tract infections in rodents despite histologic and serologic evidence of a marked immune response. To assess humoral immunity and cytokine responses in CAR bacillus disease, 6-week-old female BALB/c and C57BL/6 mice were inoculated intratracheally with 10(5) CAR bacillus organisms. CAR bacillus-specific serum immunoglobulins (immunoglobulin M [IgM], IgG1, IgG2a, IgG2b, IgG3, and IgA) and local pulmonary cytokines (tumor necrosis factor alpha [TNF-alpha], gamma interferon [IFN-gamma], and interleukin-4 [IL-4]) were evaluated by enzyme-linked immunosorbent assay every 7 days for 49 days. BALB/c mice developed CAR bacillus-induced lesions early in the course of disease that became more severe with time. Correlating with increasing disease severity, BALB/c mice had elevations in all antibody isotypes tested, and elevations in pulmonary TNF-alpha, IFN-gamma, and IL-4. C57BL/6 mice developed mild lesions with mild increases in serum IgM, IgG1, IgG2b, and IgG3 levels and minimally detectable IgG2a and IgA. Cytokine perturbations were not detected in C57BL/6 mice. The persistence of infection in BALB/c mice with vigorous serum antibody responses and increased IFN-gamma and IL-4 responses suggests that humoral immunity and T-cell responses are ineffective at preventing CAR bacillus disease. Furthermore, the lackluster antibody responses and undetectable cytokine responses in C57BL/6 mice suggest that humoral immunity and T-cell responses are not critical in resistance to CAR bacillus-induced disease.